The antisense technology in theory provides a superb method for validating gene function. However, significant problems exist in the use of the all-phosphorothioate backbone oligonucleotides. These problems result from charge, sulfur content, and non-sequence specificity. We hypothesize that reducing charge density can ameliorate these. Therefore, in Specific Aim 1 we will synthesize oligos which contain partially charged, alternating methyklphosphonate (MP)/phosphodiester (PO) and phosphorothioate (PS) backbones (alt-OMPs). Antisense alt-OMPs contain 2'-O-methylribonucleosides (alt-mr-OMPs) and are designed to interact with single-stranded RNA targets. Methods will be developed to prepare alt-mr-OMPs with MP linkages of defined configuration. Chimeric oligomers (chi-OMPs) having MP linkages at the 3' and 5'-ends of the oligomer and internal PO or PS linages will also be examined. In order to quantitate non sequence specificity, we will determine a rank order of oligo non-sequence specificity by their ability to bind to Mac-1. Comparisons will be made between two oligos, antisense c-myb, which is delivered naked, and antisense PKC-alpha (Isis 3521) which is delivered with a novel vehicle, a cationic porphyrin. We will then, in Specific Aim 2, discriminate "true" antisense at the mRNA level from toxicity by: 1) using stereoregular PS oligos because of differential nuclease cleavage of the 3' terminal PS to a nucleotide monothiophosphate: MP linkages of defined stereochemistry will also be examined; 2) We will develop a functional method to determine of alt-mr-OMPs interact with their mRNA targets in living cells. The procedure will use backbone- optimized, biotinylated, psoralen-derivatized alt-mr-OMPs which are designed to form a covalent adduct with mRNA. The resulting restriction fragment which is attached to the alt-mr-OMP will be captured on streptavidin beads. The captured fragment will be amplified by PCR using a set of specific primers. 3) Determining if the c-myb oligo has higher order structure, and leads to alterations in the rate of endosomal efflux. We will correlate this with the ability of Specific Aim 1 oligos to escape from endosomes. These experiments will be performed by a confocal microscopic technique. Finally, in Specific Aim 3, we will amplify the delivery of alt-OMPs and chi-OMPs to cells by condensing them with a novel delivery agent, tetra (N-methylpyridyl) porphine.